[0001] The invention relates to the field of environment, soil science and soil sanitation
technology. The invention particularly relates to in situ acceleration of biological
degradation of chlorinated hydrocarbons in a soil.
[0002] Chlorinated hydrocarbons are good fat solvents. Therefore these substances are frequently
used in the metal industry and in dry cleaning. Thereby, up to the 1980s, large amounts
of chlorinated hydrocarbons have ended up in the ground to severely pollute the soils
there. Removing these pollutants is difficult and complex. This is because chlorinated
hydrocarbons are heavier than water, so that they may be present in the soil to a
great depth in high concentrations. In addition, they only slowly pass into dissolution
and they thus form secondary pollution sources for long periods, which are to be sanitized
to prevent further pollution.
[0003] Currently, biological degradation of these chlorinated hydrocarbons is the most effective
manner of sanitation. In the field, here, use is made of addition of a single substance
to a soil for in situ acceleration of the biological degradation process. A drawback
that is attached to the current known methods is that non-predictable lag phases occur,
in which no conversion of chlorinated hydrocarbon is measurable. A further drawback
is that the half-life of chlorinated hydrocarbon is indeed reduced by most known methods,
but is still long. Therefore, a first object of the invention is to shorten the lag
phase of the biological degradation of chlorinated hydrocarbon. A second object is
to shorten the half-life of biological degradation of chlorinated hydrocarbon. The
objects of the invention are achieved by addition of at least H
2 (hydrogen), CO
2 (carbon dioxide) and acetate to a soil, in such a way that biological degradation
of chlorinated hydrocarbon in a soil is strongly stimulated. Then, the stimulation
is such that the lag phase of a method for stimulation of biological degradation of
chlorinated hydrocarbon becomes better predictable and is shortened compared to the
lag phases occurring with the methods known in the field. Further, by a method according
to the invention, the half-life of chlorinated hydrocarbon is shortened compared to
methods which were so far known in the field. Without wishing to be bound to any theory,
it appears that the good effect of the method according to the invention compared
to the methods used in the field can be explained as follows. It appears that in the
biostimulations currently used so far, carbon dioxide, acetate and/or hydrogen are
the limiting factors. This results in long, non-predictable lag phases. With the present
approach of the invention, a solution is found for this by artificially offering CO
2 and H
2, for instance as gases, and, dissolved or not dissolved, acetate in overmeasure.
As a result, with a method according to the invention, generally the amount of chlorinated
hydrocarbon is the rate-determining step. With a method according to the invention,
a large amount of polluting chlorinated hydrocarbon in a soil will thus result in
a high rate of conversion of chlorinated hydrocarbon. Of course, such a fast conversion
of the polluting chlorinated hydrocarbon is very desirable since, from a soil contaminated
with chlorinated hydrocarbon, its pollutants are to be removed as soon as possible
so that the soil is soon ready again for various uses.
[0004] So, the invention provides a strictly anaerobic in situ soil decontamination method
for the biological degradation of chlorinated hydrocarbons in soils, wherein, for
this biological decontamination, hydrogen and carbon dioxide and acetate are applied
to the soils from external sources for stimulation and support of the respiration
chain and growth of anaerobic bacteria so that they biologically degrade the chlorinated
hydrocarbons in an accelerated manner. The invention thus provides a strictly anaerobic
method for in situ acceleration of biological degradation of chlorinated hydrocarbons
in a soil in accordance with claim 1. Acceleration of biological degradation as used
in the invention comprises both the acceleration of biological degradation compared
to a situation in which no biological degradation took place and acceleration of biological
degradation compared to a situation in which biological degradation took place, but
to a smaller extent than desired and/or not to an optimal extent. A chlorinated hydrocarbon
as used in the invention comprises any chlorinated hydrocarbon, a substance comprising
at least one chlorine atom, at least one carbon atom and at least one hydrogen atom.
A chlorinated hydrocarbon as used in the invention may be both of natural origin and
of synthetic origin. Examples of chlorinated hydrocarbons are polychlorinated biphenyl
compounds (PCBs), chlorofluorocarbons (CFCs), chlorinated paraffins, tetrachloroethene,
trichloroethene and dichloroethenes. A soil as used in the invention comprises any
environment which is located or can be located below the surface of the earth. Preferably,
a soil as used in the invention comprises a type of soil, such as sand and peat. A
soil may, for instance, also comprise an isolated or non-isolated volume of ground
which is located above the surface of the earth, such as a lump of ground which has
been dug up in a polluted area for sanitation. In situ, as used in the invention,
means that the soil need not be moved and can be treated on location, i.e. the biological
degradation of chlorinated hydrocarbon can be stimulated on location. In situ may
be any desired location, for instance the location where a contaminated soil has been
found or the location where a dug-up contaminated soil is stored. It is possible,
to change a soil structure and/or to fragment a soil and/or to sieve it before accelerating
a biological degradation.
[0005] The addition of at least H
2, CO
2 and acetate to the soil from external sources is an essential feature of the invention.
This is because, while it is true that H
2, CO
2 and acetate may already be present in the soil, the amounts in which these three
substances are present in normal conditions do not reach the amounts needed for the
stimulation of biological degradation of chlorinated hydrocarbon in a soil as realized
by the invention. Acetate comprises any compound comprising the structure CH
3COO
-. An acetate is, for instance, the salt of acetic acid, for instance sodium acetate
(CH
3COONa). Other examples of an acetate are an acetate ester or ammonium acetate. Use
of an ammonium acetate has the advantage that, in addition to acetate, this compound
also provides the soil with nitrogen. If there is not sufficient nitrogen present
in a soil, then the acetate is thus added as ammonium acetate in a preferred embodiment
of the invention. H
2, CO
2 and acetate may be added in pure form and/or as part of more complex compounds and/or
in a mixture with other substances. In a preferred embodiment of the invention, H
2 and/or CO
2 and/or acetate are added in pure form, optionally at the same time as other substances.
That is to say that H
2 is preferably added as H
2, CO
2 is preferably added as CO
2 and acetate preferably as CH
3COO
-. H
2, CO
2 and acetate are added to the soil mentioned simultaneously or successively, as long
as these added substances are simultaneously present in the soil at least at one time.
The substances used in the invention can be added in any phase conceivable by a skilled
person, such as gaseous and/or liquid. Thus, for instance, CO
2 is added in a gaseous and/or liquid form.
[0006] Anaerobic bacteria need no or hardly no oxygen to be able to live. An anaerobic bacterium
comprises all anaerobic bacteria, so a bacterium is, for instance, obligatory anaerobic,
facultatively anaerobic or microaerobic. An obligatory anaerobic bacterium is not
capable of surviving in an oxygen-rich environment. A facultatively anaerobic bacterium
can survive both with and without oxygen. Microaerobic bacteria do need oxygen, but
a very small amount is sufficient for them. Most soils will already comprise a number
of anaerobic bacteria without intervention by means of a method according to the invention.
Optionally, to the soil mentioned, anaerobic bacteria are added. In one embodiment,
the invention provides a method according to the invention in which Dehalococcoides
bacteria have been added at least partly to this soil. The addition may be done to
a soil which comprises no or fewer anaerobic bacteria without intervention and/or
to a soil which comprises a different composition of bacteria before intervention.
The bacteria to be added may be bacteria which naturally occur in the respective soil,
but may alternatively be added to the soil from another location. This other location
may be another soil, but also any other source which can provide anaerobic bacteria.
[0007] The anaerobic bacteria used in the method according to the invention are lithotrophic
bacteria. Lithotrophic bacteria are chemolithotrophs or photolithotrophs. For the
invention, the chemolithotrophs are important. Lithotrophs are bacteria which use
inorganic substrates as an energy source. Lithotrophs consume reduced substances which
are electron donors in the process of the generation of energy by the lithotroph.
Photolithotrophs alternatively or, usually, additionally consume light to generate
energy therefrom. An electron acceptor substance then takes up the electrons. In the
case of degradation of chlorinated hydrocarbons, the chlorinated hydrocarbons will
generally serve as electron acceptors. In addition to an electron donor substance
and an electron acceptor substance, a lithotrophic bacterium needs a carbon source
for synthesis of the bacterial cell. Autolithotrophic bacteria use carbon dioxide
as a carbon source. Heterolithotrophs need other organic substances for their carbon
supply in addition to carbon dioxide.
[0008] Complicating factors for the biological degradation are the specific conditions in
which the degradation takes place and the fact that currently only one bacterial strain
is known which completely degrades chlorinated hydrocarbons to harmless end products.
The biological degradation of chlorinated hydrocarbons according to the invention
is carried out by the bacterial group Dehalococcoides. Members of the Dehalococcoides
group are currently the only known bacteria which degrade the soil pollutants tetrachloroethene,
trichloroethene, dichloroethenes and vinyl chloride to ethene and ethane. There are
other bacteria which can provide a part of the degradation process of tetrachloroethene
and trichloroethene, but not the whole degradation process. Therefore, anaerobic bacteria
according to the invention are Dehalococcoides bacteria. The Dehalococcoides bacterium
is a thin, disc-like organism without a peptidoglycan-like cell wall. The Dehalococcoides
bacterium is a member of the Green Nonsulfur Bacteria en uses chloroethenes as electron
acceptors for its anaerobic respiration. For the degradation of chlorinated hydrocarbons,
the Dehalococcoides bacteria have various reducing dehalogenases and hydrogenases
at their disposal. By means of these enzymes, Dehalococcoides ethenogenes is, for
instance, capable of converting complex chlorinated hydrocarbons into harmless substances
with the aid of hydrogen.
[0009] In the field, various techniques have been devised to make the Dehalococcoides bacteria
grow in order to expedite the sanitation of a soil contaminated with chlorinated hydrocarbons.
Firstly, various types of carbons sources are used for this and, secondly, various
types of electron donors. However, the carbon sources used in the field are often
unsuitable for Dehalococcoides bacteria. Use of these carbon sources used in the field
usually results in long, non-predictable lag phases, in which no conversion of chlorinated
hydrocarbons is measurable. Without wishing to be bound to any theory, it appears
likely that the respiration chain of the Dehalococcoides group is a precursor of the
respiration chains currently present in mitochondria and other bacteria. Due to this
limited respiration chain and the absence of essential proteins, enzymes, in the citric
acid cycle, the Dehalococcoides bacteria have difficulties with glucose, glucose-containing
products and the many intermediate substances, which can biologically be derived therefrom,
because these bacteria cannot properly convert these substances into energy via their
citric acid cycle. Therefore, these substances are not good carbon sources for the
Dehalococcoides bacteria. Conventional stimulation techniques for the biological degradation
of chlorinated hydrocarbons now use carbon sources which often have an inhibitory
action on, for instance, Dehalococcoides bacteria. The carbon sources provided can
be metabolized by Dehalococcoides bacteria; these are acetate and carbon dioxide.
In the present sanitation situation, in the method according to the invention, these
carbon sources are artificially applied, so that sufficient food is offered to the
bacteria. These proteins which the Dehalococcoides bacteria lack in the respiration
chain and the absent essential proteins of the citric acid cycle, which are normally
present in mitochondria and most bacteria, are bypassed in the present invention by
artificially adding the nutrients to the anaerobic Dehalococcoides bacteria which
can be essential to their respiration and growth. En passant, thereby, chlorinated
hydrogens are degraded to harmless end products.
[0010] As mentioned, most carbon sources currently used are not suitable and the Dehalococcoides
bacteria react thereto with long lag phases. Hydrogen is another component which is
obligatorily necessary for the Dehalococcoides bacteria. In the method according to
the invention, hydrogen is also artificially applied to the soils contaminated with
chlorinated hydrocarbons, so that, in the conversion of the chlorinated hydrocarbons
to harmless products, the pollutants and not the Dehalococcoides bacteria are the
rate-determining step to begin with. Artificially adding nutrients to soils contaminated
with chlorinated hydrocarbons in the form of hydrogen (H
2), carbon dioxide (CO
2), for instance gaseous hydrogen and gaseous or aqueous carbon dioxide, and acetate,
for instance in a solution of water, while the hydrogen is to serve as an electron
donor and the other two as carbon sources, is, on the one hand, to ensure that lithotrophic
prokaryotes, such as the Dehalococcoides group, grow exponentially in anaerobic conditions
and, on the other hand, to degrade chlorinated hydrocarbons in an accelerated manner.
Here, the chlorinated hydrocarbons serve as electron acceptors.
[0011] Bacteria are only capable of showing an optimal activity when the conditions are
favorable to these specific bacteria. One of the conditions that are important to
bacteria is the pH value of the environment. Therefore, the invention provides a method
according to the invention in which a pH value of the soil mentioned is brought within
a range which is optimal for the activity of these anaerobic bacteria. The pH value
indicates the acidity. With a method according to the invention, the pH can be brought
within the range mentioned by actively taking measures for this or, alternatively,
the pH in the soil mentioned already is and/or continues to be within this desired
range without actively taking further measures for this. An example of a substance
which can serve as a buffer component is acetate. Addition of acetate helps to prevent
a strong decrease and/or strong increase of the acidity in the soil. In an embodiment
of the invention, acetate dissolved in water is thus used to set a pH. So, when the
proportions of substances which are added to the soil are determined, the pH, the
acidity, of the soil can be taken into account. Here, care should be taken that, for
instance, the optimal pH within which the Dehalococcoides group operates is preferably
not negatively affected by carbon dioxide. Here, it should be noted that, if the carbon
dioxide is dissolved in water, bicarbonate is created which can form a buffer with
which a pH can be set. In an embodiment of the invention, carbon dioxide dissolved
in water is thus used to form a buffer and to set a pH. Further, substances which
are, for the rest, not necessary in an embodiment of the invention, can be used to
maintain the acidity of the soil within a desired range. A skilled person knows many
methods and substances to influence the acidity of a soil.
[0012] The pH range that is optimal for the activity of the anaerobic bacteria mentioned
will depend on the specific properties of these bacteria, and therefore on the type
of anaerobic bacteria. For most bacteria, the pH range for an optimal activity is
known and, thus, a skilled person can simply look up the right range for a specific
bacterium. Many bacteria show good activity at a more or less neutral pH. In that
case, the pH of the soil mentioned is preferably brought within a range of pH 6.5-7.5.
An example of anaerobic bacteria which show optimal activity at a more or less neutral
pH are Dehalococcoides bacteria. Therefore, in a preferred embodiment, the invention
provides a method in which the range mentioned of the pH value is 6.5-7.5, and in
which the anaerobic bacteria mentioned are Dehalococcoides bacteria.
[0013] One of the conditions which is, in addition to the pH value of the environment, important
to bacteria for them to be able to carry out an optimal activity is the temperature
of the environment. Therefore, in one embodiment, the invention provides a method
according to the invention in which the temperature of the soil mentioned is brought
within a range which is optimal for the activity of the Dehalococcoides bacteria.
The temperature can be brought within the range mentioned by actively taking measures
for this or, alternatively, the temperature in the soil mentioned already is and/or
continues to be within the desired range mentioned with a method according to the
invention without actively taking further measures for this. A skilled person knows
many methods for influencing the temperature of a soil. The temperature of the soil
may, for instance, be increased by adding hot gases and/or liquids to the soil and/or
using heat mats, a hot addition being an addition which has a temperature which is
higher than the temperature of the soil. Conversely, the temperature of the soil may,
for instance, be decreased by adding cold gases and/or liquids to the soil, a cold
addition being an addition which has a temperature which is lower than the temperature
of the soil.
[0014] The temperature range which is optimal for the activity of the anaerobic bacteria
mentioned will depend on the specific properties of these bacteria, and therefore
on the type of anaerobic bacteria. For most bacteria, the temperature range for an
optimal activity is known and, thus, a skilled person can simply look up the right
range for a specific bacterium. Many bacteria show good activity at a temperature
higher than 4°C, often particularly higher than 10°C, and lower than 40°C. In that
case, the temperature of the soil mentioned is preferably brought within a temperature
range of 4°C-40°C, more preferably within a temperature range of 10°C-40°C.. An example
of anaerobic bacteria which show optimal activity at a temperature range of 4°C-40°C,
particularly within a temperature range of 10°C-40°C, are Dehalococcoides bacteria.
Therefore, in a preferred embodiment, the invention provides a method in which the
range mentioned is 4°C-40°C, and in which the anaerobic bacteria mentioned are Dehalococcoides
bacteria. In the right conditions, such as pH and temperature, and with the right
food, such as the substances which are added according to the invention, the Dehalococcoides
bacteria degrade the chlorinated hydrocarbons well and fast.
[0015] In normal conditions, at room temperature and normal outside temperatures and at
normal atmospheric pressure, hydrogen and carbon dioxide are gaseous. So, it is simple
and inexpensive to use hydrogen and carbon dioxide in gaseous form. Therefore, in
a preferred embodiment of the invention, hydrogen and carbon dioxide are used in gaseous
form. For the same reasons of simplicity and price, acetate is preferably used when
dissolved in water. In a preferred embodiment, the invention therefore provides a
method according to the invention in which the H
2 and CO
2 mentioned are gaseous and in which the acetate mentioned is dissolved in water. This
water comprises any aqueous solution. In a preferred embodiment of the invention,
acetate comprises CH
3COO
- dissolved in water.
[0016] Up to now, in the field, if a substance was added to a soil for in situ acceleration
of the biological degradation process, the respective substance was added in a small
amount. The single addition was usually at most of the order of a few micromoles.
A method according to the invention does not only use multiple necessary substances
H
2, CO
2 and acetate, but also applies each separate substance to the soil in a surprisingly
large amount. Although a lower concentration is possible and is within the scope of
the invention, a substance according to the invention is preferably applied until
groundwater and/or soil are saturated and/or up to a concentration of at least 10
µmol/l of groundwater and/or soil, more preferably up to a concentration of at least
0.1 mmol/l of groundwater and/or soil, more preferably up to a concentration of at
least 1 mmol/l of groundwater and/or soil. In a preferred embodiment, the invention
therefore provides a method according to the invention in which the H
2, CO
2 and acetate mentioned are added until the soil mentioned and/or groundwater in the
soil mentioned is saturated and/or up to a concentration of at least 10 µmol/l of
groundwater and/or soil. The amounts of H
2, CO
2 and acetate to be added and the concentrations to be reached of course partly depend
on the situation. If the substances are, for instance, added to sanitize a pollution
source, the amounts to be added which often be larger than if the substances are added
to sanitize the associated plume, because the concentrations of chlorinated hydrocarbons
in the pollution source will usually be higher than in the plume. In an embodiment,
in a source, preferably concentrations of H
2, CO
2 and acetate of at least 1 mmol/l of groundwater or soil are reached. In a plume,
the concentration of hydrocarbon will usually be lower than in the source and lower
concentrations of H
2, CO
2 and acetate will be sufficient. Since H
2, CO
2 and acetates are slightly active even with very low concentrations of, for instance,
tenths of micromoles per liter, additions which result in low concentrations in the
soil are within the scope of the invention.
[0017] In a preferred embodiment according to the invention, in addition to H
2, CO
2 and acetate, still other substances are added to a soil. The other substances which
can be added further may be all substances a skilled person would like to add further.
The substances according to the invention may be added in any phase conceivable by
a skilled person, such as for instance gaseous and/or liquid. In an embodiment the
other substances are preferably nutrients for anaerobic bacteria. These nutrients
for anaerobic bacteria are all substances which play a role in the life of these anaerobic
bacteria. Examples of substances which form important nutrients for many anaerobic
bacteria are nitrogen, phosphate and magnesium. In one embodiment, the invention therefore
provides a method according to the invention in which, to the soil mentioned, further,
the substances phosphate and/or nitrogen and/or magnesium are added. Phosphate and/or
nitrogen and/or magnesium and/or other substances which can be added to a soil in
a method according to the invention may be added in any form, for instance pure substances
and/or simple compounds and/or complex compounds and/or mixtures, as long as the respective
form is a source of the respective substance for anaerobic bacteria.
[0018] Addition of substances to a soil can take place in all possible manners. A skilled
person knows many methods for adding substances to a soil. A soil is, for instance,
added to a soil contaminated with chlorinated hydrocarbon with the aid of injections
or other aids. Any manner of addition is suitable as long as the added substance reaches
the area contaminated with chlorinated hydrocarbon and preferably reaches a concentration
of at least 10 µmol/l, more preferably of at least 0.1 mmol/l, most preferably of
at least 1 mmol/l. An example of a suitable manner of adding substances according
to a method of the invention is injecting substances. Substances which are added in
gaseous form are, in such a method, preferably injected below an area comprising chlorinated
hydrocarbon and/or into an area comprising chlorinated hydrocarbon. Substances which
are added in liquid and/or dissolved form are, in such a method, preferably injected
into an area comprising this chlorinated hydrocarbon and/or above an area comprising
this chlorinated hydrocarbon. In one embodiment, the invention therefore provides
a method according to the invention in which, in the soil mentioned, above-mentioned
gaseous H
2 and/or CO
2 and any other gaseous substances to be added are at least injected below an area
comprising this chlorinated hydrocarbon and in which above-mentioned acetate and any
other liquid and/or dissolved substances to be added are at least injected into an
area comprising this chlorinated hydrocarbon. In methods as used in the field, what
it usually comes down to is that the gaseous nutrients are injected far below the
pollution source and plume and that the gases then automatically diffuse upwards.
On their way up, these gases encounter those bacteria that need these gases for the
respiration and growth. En passant, thereby, chlorinated hydrocarbons are converted
into harmless substances. The necessary aqueous substances are usually injected into
the source and plume. The gaseous substances added are preferably added at a pressure
which does not differ too much from a pressure in the soil to prevent the gases from
escaping at the surface without first saturating the soil.
[0019] The biostimulation, gaseous in a preferred embodiment, which is carried out artificially
in combination with application of some other bacterial nutrients to the soil, serves
the purpose of sanitizing soils contaminated with chlorinated hydrocarbons in an accelerated
manner at strongly reduced costs compared to conventional sanitation methodologies.
The carbon dioxide supplemented by acetate, for instance dissolved in water, and further,
for instance, a phosphate and a nitrogen source, are responsible for the growth of
the anaerobic bacteria. Both gases, H
2 and CO
2, in any possible proportion to each other, optionally diluted with gaseous nitrogen,
another gas and other nutrients in combination with acetate dissolved in water, phosphate
and a nitrogen source, are simultaneously or each separately added to the contaminated
soils with the aid of injections or other aids, while they are offered there to the
Dehalococcoides bacteria, in overmeasure. Accordingly, after a short incubation, stimulation
and a growth period of the anaerobic bacteria, not the number of bacteria per volume
unit, but particularly the amount of electron acceptors in the form of chlorinated
hydrocarbons is the rate-determining step in the further growth of the bacteria.
[0020] Because of the lithotrophic properties of the Dehalococcoides bacteria, reduction
of the costs, simplicity and convenience of the present method, in an embodiment of
the invention, a deliberate choice was made for acetate dissolved in water as a primary
carbon source in combination with gaseous carbon dioxide (CO
2) as a secondary carbon source. With acetate as a primary carbon source in addition
to sufficient Dehalococcoides bacteria, per volume unit, and hydrogen as an electron
donor, half-lives for chlorinated hydrocarbons in polluted soils of a few hours can
be achieved. Here, Dehalococcoides bacteria show the best activity in strictly anaerobic
conditions, where hydrogen acts as an electron carrier, carbon dioxide and acetate
as carbon sources and chlorinated hydrocarbons as electron acceptors. Since a method
according to the invention is for accelerating biological degradation of chlorinated
hydrocarbons in a soil by means of Dehalococcoides bacteria and Dehalococcoides bacteria
are capable of degrading tetrachloroethene (also called perchloroethene) and trichloroethene
to ethene and ethane, in an embodiment of the invention, chlorinated hydrocarbon comprises
inter alia tetrachloroethene and trichloroethene. Dehalococcoides bacteria can also degrade,
for instance, dichloroethene, vinyl chloride and a polychlorinated biphenyl compound
to ethene and ethane. In one embodiment, the invention therefore provides a method
according to the invention for in situ acceleration of biological degradation of chlorinated
hydrocarbons in a soil, in which this chlorinated hydrocarbon is tetrachloroethene
and/or thrichloroethene and/or a dichloroethene and/or vinyl chloride and/or a polychlorinated
biphenyl compound.
[0021] In a further aspect, the invention provides use of H
2, CO
2 and acetate for strictly anaerobic in situ biological degradation or in situ acceleration
of biological degradation of chlorinated hydrocarbons in a soil in accordance with
claim 12. These substances may be used in any concentration and in any phase. Preferably,
H
2 is used as pure H
2 under pressure, CO
2 is used as pure CO
2 under pressure and/or in a liquid form and acetate is used as acetate in an aqueous
solution. In addition to H
2, CO
2 and acetate, any other substance may be used, such as for instance one or more auxiliary
substances. Preferably, nutrients for anaerobic bacteria, such as phosphate and/or
nitrogen and/or magnesium are used.
[0022] Artificially adding nutrients to soils contaminated with chlorinated hydrocarbons
in the form of gaseous hydrogen (H
2) and gaseous carbon dioxide (CO
2), as an electron donor and a carbon source, respectively, is, on the one hand, to
ensure that lithotrophic prokaryotes, such as the Dehalococcoides group, exponentially
grow in anaerobic conditions and, on the other hand, to degrade chlorinated hydrocarbons
in an accelerated manner. Here, these chlorinated hydrocarbons serve as electron acceptors.
The carbon dioxide supplemented by acetate dissolved in water, phosphate and a nitrogen
source is responsible for the growth of the anaerobic bacteria.
[0023] This gaseous biostimulation, which is artificially applied to the soils in combination
with some other bacterial nutrients, serves the purpose of sanitizing soils contaminated
with chlorinated hydrocarbons in an accelerated manner at strongly reduced costs compared
to conventional sanitation methodologies.
[0024] Both gases, in any possible proportion to each other, optionally diluted with gaseous
nitrogen, another gas and other nutrients in combination with acetate dissolved in
water, phosphate and a nitrogen source, are simultaneously or each separately added
to the contaminated soils with the aid of injections or other aids, where they are
offered to the Dehalococcoides bacteria, in overmeasure. Accordingly, after a short
incubation, stimulation and a growth period of the anaerobic bacteria, not the number
of bacteria per volume unit, but particularly the amount of electron acceptors in
the form of chlorinated hydrocarbons is the rate-determining step in the further growth
of the bacteria.
[0025] With acetate as a primary carbon source in addition to sufficient Dehalococcoides
bacteria, per volume unit, and hydrogen as an electron donor, half-lives for chlorinated
hydrocarbons in polluted soils of a few hours can be achieved.
[0026] Because of the lithotrophic properties of the Dehalococcoides bacteria, reduction
of the costs, simplicity and convenience of the present method, in an embodiment of
the invention, a deliberate choice was made for acetate dissolved in water as a primary
carbon source in combination with gaseous carbon dioxide (CO
2) as a secondary carbon source.
[0027] Members of the Dehalococcoides group are currently the only known bacteria which
degrade the soil pollutants tetrachloroethene and trichloroethene to ethene and ethane.
For this, the Dehalococcoides bacteria have various reducing dehalogenases and hydrogenases
at their disposal. By means of these enzymes, Dehalococcoides ethenogenes is, for
instance, capable of converting complex chlorinated hydrocarbons into harmless substances
with the aid of hydrogen.
[0028] The Dehalococcoides bacterium is a thin, disc-like organism without a peptidoglycan-like
cell wall. The Dehalococcoides bacterium is a member of the Green Nonsulfur Bacteria
en uses chloroethenes as electron acceptors for its anaerobic respiration.
[0029] It is likely that the respiration chain of the Dehalococcoides group is a precursor
of the respiration chains currently present in mitochondria and other bacteria. Due
to this limited respiration chain and the absence of essential enzymes in the citric
acid cycle, the Dehalococcoides bacteria have difficulties with glucose, glucose-containing
products and the many intermediate substances, which can biologically be derived therefrom.
Therefore, these substances are not good carbon sources for the Dehalococcoides bacteria.
[0030] Use of these carbon sources usually results in long, non-predictable lag phases,
in which no conversion of chlorinated hydrocarbons is measurable.
[0031] The carbon sources which can be metabolized by Dehalococcoides bacteria are acetate
and carbon dioxide.
[0032] Chlorinated hydrocarbons are good fat solvents. Therefore these substances are frequently
used in the metal industry and in dry cleaning. Thereby, up to the 1980s, large amounts
of chlorinated hydrocarbons have ended up in the ground to severely pollute the soils
there. Removing these pollutants is difficult and complex.
[0033] This is because chlorinated hydrocarbons are heavier than water, so that they may
be present in the soil to a great depth in high concentrations. In addition, they
only slowly pass into dissolution and they thus form secondary pollution sources for
long periods, which are to be sanitized to prevent further pollution.
[0034] Currently, biological degradation of these chlorinated hydrocarbons is the most effective
manner of sanitation. This biological degradation is carried out by the bacterial
group Dehalococcoides. As stated, this is done in strictly anaerobic conditions with
hydrogen as an electron carrier, carbon dioxide and acetate as carbon sources and
chlorinated hydrocarbons as electron acceptors.
[0035] In the right conditions, such as pH and temperature, and with the right food, the
Dehalococcoides bacteria degrade the chlorinated hydrocarbons well and fast.
[0036] Complicating factors for this biological degradation are the specific conditions
in which the degradation takes place and the fact that currently only one bacterial
strain is known which completely degrades chlorinated hydrocarbons to harmless end
products.
[0037] In order to expedite the sanitation of chlorinated hydrocarbons, various techniques
have been devised to make this bacterial strain grow. Firstly, various types of carbons
sources are used for this and, secondly, various types of electron donors.
[0038] As previously mentioned, most carbon sources currently used are unsuitable and Dehalococcoides
bacteria react thereto with long lag phases.
[0039] Dehalococcoides bacteria are lithotrophic, that is to say that carbon dioxide is
one of their carbon sources. The other carbon source is acetate. In the present sanitation
situation, these carbon sources are artificially applied, so that sufficient food
is offered to the bacteria. Here, care should be taken that the optimal pH within
which the Dehalococcoides group operates is not negatively affected by the carbon
dioxide.
[0040] Hydrogen is another component which is obligatorily necessary for the Dehalococcoides
bacteria. This gas is also artificially applied to the soils contaminated with chlorinated
hydrocarbons, so that, in the conversion of the chlorinated hydrocarbons to harmless
products, the pollutants and not the Dehalococcoides bacteria are the rate-determining
step to begin with.
[0041] In the biostimulations currently used, carbon dioxide, acetate and/or hydrogen are
the limiting factors. This results in long, non-predictable lag phases. With the present
approach of the invention, a solution is found for this by artificially offering both
gases and dissolved acetate in overmeasure. What it usually comes down to is that
the gaseous nutrients are injected far below the pollution source and plume and that
the gases then automatically diffuse upwards. On their way up, these gases encounter
bacteria which need these gases for the respiration and growth. En passant, thereby,
chlorinated hydrocarbons are converted into harmless substances. The necessary aqueous
substances are usually injected into the source and plume.
[0042] All techniques which serve to contact the gaseous nutrients hydrogen and carbon dioxide
and the aqueous acetate solution with Dehalococcoides bacteria in a safe and responsible
manner, so that they can use the chlorinated hydrocarbons for their respiration, are
covered by this patent.
[0043] In one embodiment, the invention provides a strictly anaerobic in situ soil decontamination
method for the biological degradation of chlorinated hydrocarbons in soils, wherein,
for this biological decontamination, gas mixtures consisting of hydrogen and carbon
dioxide and acetate dissolved in water are applied to the soils from external sources
for stimulation and support of the respiration chain and growth of the Dehalococcoides
bacterial group, so that they biologically degrade the chlorinated hydrocarbons in
an accelerated manner. In a further embodiment, the invention provides that above-mentioned
external application used of gas mixtures consisting of hydrogen and carbon dioxide
and acetate dissolved in water also holds for all soils contaminated with chlorinated
hydrocarbons, where the respective bacteria have been added to these soils from another
location. Above-mentioned gas mixtures used of hydrogen and carbon dioxide can be
added to the soil each separately or simultaneously in any conceivable proportion,
optionally diluted with other gases and auxiliary substances, but in any case simultaneously
or separately with acetate dissolved in water in a safe, efficient and adequate manner
with the aid of any aid suitable for this. Further, above-mentioned gas mixtures and
liquids used may be applied to the soils contaminated with chlorinated hydrocarbons
in one go or gradually at any conceivable rate, amount and frequency, while the location
and depth of the gas mixtures to be used depends on the location of the pollution,
so that an optimal biostimulation for the degradation of the chlorinated hydrocarbons
is created. Further, any aid and form of application for applying the above-mentioned
nutrients to the contaminated soils falls within the scope of this invention.
[0044] The invention relates to the in situ biostimulation of Dehalococcoides bacteria,
with the aid of gaseous nutrients, such as hydrogen and carbon dioxide in combination
with acetate dissolved in water in soils contaminated with chlorinated hydrocarbons.
These nutrients are applied to these soils, for the purpose of biologically sanitizing
these soils in an accelerated and cost-effective manner.
[0045] Currently, Dehalococcoides bacteria are the only prokaryotes which degrade tetrachloroethene
and trichloroethene to ethene. For this, these bacteria have various dehalogenases
and hydrogenases at their disposal. Here, hydrogen serves as an electron donor. The
carbon sources for these Dehalococcoides bacteria are carbon dioxide and acetate.
For their anaerobic respiration, they exclusively use chlorinated hydrocarbons.
[0046] The gaseous nutrients and nutrients dissolved in water which are artificially used
for biostimulation of the Dehalococcoides group, can safely and adequately be applied
to the soils contaminated with chlorinated hydrocarbons in any possible proportion,
rate and amount, in such a manner that an optimal biostimulation is created.
[0047] Conventional stimulation techniques for the biological degradation of chlorinated
hydrocarbons now use carbon sources which often have an inhibitory action on, for
instance, Dehalococcoides bacteria, because these bacteria cannot convert these substances
into energy via their citric acid cycle. They lack essential proteins for this, which
are normally present in mitochondria and most bacteria, in the respiration chain and
the citric acid cycle,.
[0048] In the present invention, these proteins are bypassed by artificially adding nutrients
to the anaerobic Dehalococcoides bacteria, which are essential to their respiration
and growth. En passant, here, chlorinated hydrogens are degraded to harmless end products.
1. A strictly anaerobic method for in situ acceleration of biological degradation of
chlorinated hydrocarbons in a soil by Dehalococcoides bacteria, comprising adding
to said soil simultaneously or successively from external sources at least the following
substances: H2, CO2 and acetate, whereby hydrogen is an electron carrier, carbon dioxide and acetate
are carbon sources and the chlorinated hydrocarbons are electron acceptors, wherein
said soil comprises Dehalococcoides bacteria, and wherein said substances are simultaneously
present in the soil.
2. A method according to claim 1, wherein said Dehalococcoides bacteria have at least
partly been added to said soil.
3. A method according to any one of claims 1-2, wherein a pH value of said soil is brought
within a range which is optimal for the activity of said Dehalococcoides bacteria.
4. A method according to claim 3, wherein said range is pH value 6.5-7.5.
5. A method according to any one of claims 1-4, wherein the temperature of said soil
is brought within a range which is optimal for the activity of said Dehalococcoides
bacteria.
6. A method according to claim 5, wherein said range is 4°C-40°C.
7. A method according to any one of claims 1-6, wherein said H2 is gaseous, wherein said acetate is dissolved in water and wherein said CO2 is gaseous or dissolved in water.
8. A method according to any one of claims 1-7, wherein said H2, CO2 and acetate are added up to a concentration of at least 10 µmol/1 of groundwater
or soil.
9. A method according to any one of claims 1-8, wherein, to said soil, further, the substances
phosphate and/or nitrogen and/or magnesium are added.
10. A method according to any one of claims 1-9, wherein, in said soil, from external
sources gaseous H2 and/or CO2 and any other gaseous substances to be added are at least injected below an area
comprising said chlorinated hydrocarbon and wherein said acetate and any other liquid
and/or dissolved substances to be added are at least injected into an area comprising
said chlorinated hydrocarbon.
11. A method according to any one of claims 1-10, wherein said chlorinated hydrocarbon
is tetrachloroethene and/or trichloroethene and/or a dichloroethene and/or vinyl chloride
and/or a polychlorinated biphenyl compound.
12. Use of H2, CO2 and acetate for strictly anaerobic in situ acceleration of biological degradation
of chlorinated hydrocarbons in a soil by Dehalococcoides bacteria, whereby hydrogen
is an electron carrier, carbon dioxide and acetate are carbon sources and the chlorinated
hydrocarbons are electron acceptors, wherein H2, CO2 and acetate are added simultaneously or successively to said soil from external sources,
and wherein said soil comprises Dehalococcoides bacteria.
1. Streng anaerobes Verfahren zur in-situ-Beschleunigung biologischen Abbaus von Chlorkohlenwasserstoffen
in einem Boden durch Dehalococcoides-Bakterien, umfassend, dass dem Boden gleichzeitig
oder nacheinander aus externen Quellen mindestens die folgenden Substanzen zugesetzt
werden: H2, CO2 und Acetat, wobei Wasserstoff ein Elektronenträger ist, Kohlendioxid und Acetat Kohlenstoffquellen
sind und die Chlorkohlenwasserstoffe Elektronenakzeptoren sind, wobei der Boden Dehalococcoides-Bakterien
umfasst und wobei die Substanzen gleichzeitig im Boden vorhanden sind.
2. Verfahren nach Anspruch 1, wobei die Dehalococcoides-Bakterien mindestens teilweise
dem Boden zugesetzt worden sind.
3. Verfahren nach einem der Ansprüche 1-2, wobei ein pH-Wert des Bodens in einen Bereich
gebracht wird, der optimal für die Aktivität der Dehalococcoides-Bakterien ist.
4. Verfahren nach Anspruch 3, wobei der Bereich des pH-Werts 6,5-7,5 ist.
5. Verfahren nach einem der Ansprüche 1-4, wobei die Temperatur des Bodens in einen Bereich
gebracht wird, der optimal für die Aktivität der Dehalococcoides-Bakterien ist.
6. Verfahren nach Anspruch 5, wobei der Bereich 4°C-40°C ist.
7. Verfahren nach einem der Ansprüche 1-6, wobei das H2 gasförmig ist, wobei das Acetat in Wasser gelöst ist und wobei das CO2 gasförmig oder in Wasser gelöst ist.
8. Verfahren nach einem der Ansprüche 1-7, wobei das H2, CO2 und Acetat bis zu einer Konzentration von mindestens 10 µmol/l Grundwasser oder Boden
zugesetzt werden.
9. Verfahren nach einem der Ansprüche 1-8, wobei dem Boden ferner die Substanzen Phosphat
und/oder Stickstoff und/oder Magnesium zugesetzt werden.
10. Verfahren nach einem der Ansprüche 1-9, wobei in den Boden gasförmiges H2 und/oder CO2 und alle anderen gasförmigen, zuzusetzenden Substanzen mindestens unter einen den
Chlorkohlenwasserstoff umfassenden Bereich eingespritzt werden, und wobei das Acetat
und alle anderen zuzusetzenden flüssigen und/oder gelösten Substanzen mindestens in
einen den Chlorkohlenwasserstoff umfassenden Bereich eingespritzt werden.
11. Verfahren nach einem der Ansprüche 1-10, wobei der Chlorkohlenwasserstoff Tetrachlorethen
und/oder Trichlorethen und/oder Dichlorethen und/oder Vinylchlorid und/oder eine polychlorierte
Biphenylverbindung ist.
12. Verwendung von H2, CO2 und Acetat für eine streng anaerobe in-situ-Beschleunigung biologischen Abbaus von
Chlorkohlenwasserstoffen in einem Boden durch Dehalococcoides-Bakterien, wobei Wasserstoff
ein Elektronenträger ist, Kohlendioxid und Acetat Kohlenstoffquellen sind und die
Chlorkohlenwasserstoffe Elektronenakzeptoren sind, wobei H2, CO2 und Acetat gleichzeitig oder nacheinander dem Boden aus externen Quellen zugesetzt
werden und wobei der Boden Dehalococcoides-Bakterien umfasst.
1. Procédé strictement anaérobie destiné à accélérer in situ la dégradation biologique d'hydrocarbures chlorés dans un sol par des bactéries Dehalococcoides,
comprenant l'addition audit sol simultanément ou successivement à partir de sources
externes d'au moins les substances suivantes : H2, CO2 et acétate, moyennant quoi l'hydrogène est un porteur d'électrons, le dioxyde de
carbone et l'acétate sont des sources de carbone et les hydrocarbures chlorés sont
des accepteurs d'électrons, dans lequel ledit sol comprend des bactéries Dehalococcoides,
et dans lequel lesdites substances sont simultanément présentes dans le sol.
2. Procédé selon la revendication 1, dans lequel lesdites bactéries Dehalococcoides ont
été au moins partiellement ajoutées audit sol.
3. Procédé selon l'une quelconque des revendications 1 à 2, dans lequel une valeur de
pH dudit sol est amenée dans une plage qui est optimale pour l'activité desdites bactéries
Dehalococcoides.
4. Procédé selon la revendication 3, dans lequel ladite plage est une valeur de pH de
6,5 à 7,5.
5. Procédé selon l'une quelconque des revendications 1 à 4, dans lequel la température
dudit sol est amenée dans une plage qui est optimale pour l'activité desdites bactéries
Dehalococcoides.
6. Procédé selon la revendication 5, dans lequel ladite plage est de 4 °C à 40 °C.
7. Procédé selon l'une quelconque des revendications 1 à 6, dans lequel ledit H2 est gazeux, dans lequel ledit acétate est dissous dans l'eau et dans lequel ledit
CO2 est gazeux ou dissous dans l'eau.
8. Procédé selon l'une quelconque des revendications 1 à 7, dans lequel lesdits H2, CO2 et acétate sont ajoutés à une concentration d'au moins 10 µmol/l d'eau souterraine
ou de sol.
9. Procédé selon l'une quelconque des revendications 1 à 8, dans lequel, audit sol, en
outre, les substances phosphate et/ou azote et/ou magnésium sont ajoutées.
10. Procédé selon l'une quelconque des revendications 1 à 9, dans lequel, dans ledit sol,
le H2 et/ou le CO2 gazeux et toute autre substance gazeuse à ajouter sont au moins injectés sous une
zone comprenant ledit hydrocarbure chloré et dans lequel ledit acétate et toute autre
substance liquide et/ou dissoute à ajouter sont au moins injectés dans une zone comprenant
ledit hydrocarbure chloré.
11. Procédé selon l'une quelconque des revendications 1 à 10, dans lequel ledit hydrocarbure
chloré est du tétrachloroéthène et/ou du trichloroéthène et/ou un dichloroéthène et/ou
du chlorure de vinyle et/ou un composé biphényle polychloré.
12. Utilisation de H2, de CO2 et d'acétate pour l'accélération in situ strictement anaérobie de la dégradation biologique d'hydrocarbures chlorés dans un
sol par des bactéries Dehalococcoides, moyennant quoi l'hydrogène est un porteur d'électrons,
le dioxyde de carbone et l'acétate sont des sources de carbone et les hydrocarbures
chlorés sont des accepteurs d'électrons, dans laquelle le H2, le CO2 et l'acétate sont ajoutés simultanément ou successivement audit sol à partir de sources
externes, et dans laquelle ledit sol comprend des bactéries Dehalococcoides.